This invention relates to the field of pharmaceutically active or otherwise beneficial compounds obtained from natural sources. In particular, the invention provides a seed preparation enriched in phenethyl isothiocyanate, a naturally-occurring anticancer and cancer preventative substance.
Various scientific articles are referred to in parentheses throughout the specification, and complete citations are listed at the end of the specification. These articles are incorporated by reference herein to describe the state of the art to which this invention pertains.
Most people are familiar with the biting taste of horseradish and mustard, the pungent flavors of cabbage and Brussels sprouts and the peppery sensation of watercress. These plants belong to a broad group of species consisting of the Cruciferae and fourteen other families, which contain over 100 related natural chemical compounds, called glucosinolates, which are responsible for the familiar flavors and aromas of these plants. Plants that contain glucosinolates are widely consumed by people and livestock. The occurrence and biochemistry of glucosinolates has been well-documented. The variation in glucosinolate content among these plants is tremendous. Some plants can contain predominantly one form of glucosinolate while others are characterized by as many as fifteen different forms.
Glucosinolates are nitrogenous natural products that are derived from one of several different amino acids. Glucosinolates also contain sulfur from cysteine as well as a molecule of glucose, which is attached by a thioglucosidic bond. Many plants contain very high concentrations of glucosinolates, which presumably serve a protective function (Mithen, 1992). When plant tissues are disrupted, the glucosinolates rapidly break down into one of several forms. The first step of this breakdown is catalyzed by a class of enzymes generally referred to as myrosinases. The unstable aglycone which results from the removal of the glucose moiety by the myrosinase then rearranges into one of three basic forms by a process which is generally spontaneous. The basic forms that result from this rearrangement are either isothiocyanates, nitrites or thiocyanates. The wide variety of forms of glucosinolates and their breakdown products results from a biosynthetic pathway that originates from different amino acids, followed by subsequent modifications, all of which seem to be species specific. Although glucosinolates have been the focus of intensive research, many aspects of this diverse biochemical system have yet to be resolved.
Vegetables that contain glucosinolates have long been known to be a healthy part of the daily diet. For instance, the isothiocyante, sulforaphane, has been shown to be a powerful cancer preventive compound that specifically induces phase II detoxification enzymes (Zhang et al., 1992). Sulphoraphane is one example of several isothiocyanates that are characterized by similar kinds of health benefits. PEITC (phenethyl iosthiocyanate) is a glucosinolate breakdown product which is similar to sulforaphane and has also been a focus of intensive cancer preventive research. In addition to the extensive research done with animal systems, PEITC from fresh watercress has been shown to specifically inhibit the oxidation of nitrosamines from tobacco in human smokers as measured by urinary excretion of metabolites (Hecht et al., 1995). PEITC has been repeatedly shown to be both an effective and stable cancer preventive and anticancer compound. Not only does it inhibit the carcinogenic activation of many of the components of tobacco products, but prevents similar effects of many other toxins as well as even promote the death of cancerous cells.
The health promoting and anticancer benefits of PEITC may be obtained by consuming large amounts of the vegetables that are rich in this substance. However, such consumption may not be practical or desirable. It would be preferable if PEITC could be obtained in a more concentrated form such that its benefits could be enjoyed, for instance, through daily consumption of a small capsule, rather than large amounts of PEITC-containing vegetables.
In accordance with the present invention, plant varieties and specific tissues have been identified that are rich natural sources of PEITC, and methods have been devised to increase the production of PEITC in these tissues and to obtain preparations of certain plant tissues that are highly enriched in PEITC.
According to one aspect of the present invention, a preparation of disrupted plant tissue, comprising at least 1 mg PEITC per gram fresh weight of the plant tissue, is provided. Preferably, the preparation comprises at least 5 mg PEITC per gram fresh weight plant tissue, and most preferably at least 10 mg PEITC per gram fresh weight plant tissue. In a preferred embodiment, the preparation is made from seeds of upland cress, and may be provided as a dried product.
According to another aspect of the invention, a nutraceutical formulation is provided, which comprises the plant tissue preparation described above.
A preferred embodiment of the present invention provides a crushed, dried preparation of upland cress seed, comprising at least 5 mg PEITC per gram dry weight. A nutraceutical formulation also provided, comprising this preparation.
According to another aspect of the present invention, a method is provided for obtaining a plant tissue preparation that contains at least 1 mg PEITC per gram fresh weight of the tissue. The method comprises: (a) providing fresh or fresh-frozen plant tissue; (b) disrupting the tissue; and (c) incubating the disrupted tissue in an aqueous solution for a time and at a temperature effective to produce the preparation that contains at least 1 mg PEITC per gram fresh weight of the tissue. In one embodiment, the disrupted tissue is incubated in water, a method preferred when the tissue is incubated at slightly elevated temperature, e.g., 30xc2x0 C. In another embodiment, the disrupted tissue is incubated in a biologically compatible buffer. Preferably, the pH of the disrupted tissue in the aqueous solution is between 4.0 and 8.0, more preferably between 4.5 and 7.2.
In the aforementioned method, the incubation is performed at a temperature between 20xc2x0 C. and 37xc2x0 C., more preferably between 27xc2x0 C. and 32xc2x0 C., and most preferably at 30xc2x0 C. The incubation is performed for at least 2 minutes and preferably between 10 and 40 minutes, most preferably for 20 minutes.
It is preferred that the aforementioned method be practiced on upland cress seeds. It is also preferred that the seeds are frozen in liquid nitrogen before disruption. After disruption, the plant material may be subjected to freeze-drying, preferably to a final temperature of less than 10xc2x0 C., more preferably to 0xc2x0 C.
According to another aspect of the invention, a plant tissue preparation comprising at least 1 mg PEITC per gram fresh weight plant tissue, prepared by the aforementioned method, is provided.
According to a specific aspect of the present invention, a method of obtaining a preparation of upland cress seed containing PEITC is provided. The method comprises: (a) providing fresh or fresh-frozen upland cress seed; (b) crushing the seeds; and (c) incubating the seeds in an aqueous solution at 30xc2x0 C. for 20 minutes. The method may further comprise freeze-drying the preparation to a final temperature of 0xc2x0 C. A PEITC-containing upland cress seed preparation, prepared by the aforementioned method, is also provided, as is a nutraceutical formulation for prevention or treatment of cancer, which comprises the upland cress seed preparation.
Other features and advantages of the present invention will be better understood by reference to the drawings, detailed description and examples that follow.